High strength intraosseous implants

ABSTRACT

The present invention enables modification of an intraosseous implant device that is not only biologically non-inert, but can stimulate bone and vascular growth; decrease localized inflammation; and fight local infections. The method of the present invention provides a fiber with any of the following modifications: (1) Nanofiber with PDGF, (2) Nanofiber with PDGF+BMP2, and (3) Nanofiber with BMP2 and Ag. Nanofiber can be modified with other growth factors that have been shown to improve bone growth and maturation—BMP and PDGF being the most common. Nanofiber can be applied on the surface of the implant in several ways. First, a spiral micro-notching can be applied on the implant in the same direction as the threads with the nanofibers embedded into the notches. Second, the entire surface of the implant may be coated with a mesh of nanofibers. Third, it can be a combination of both embedding and notching.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/374,700 filed Jul. 13, 2021 by the University of Central Oklahomaentitled “High strength intraosseous implants” which is a continuationof U.S. patent application Ser. No. 16/286,005 filed Feb. 26, 2019 bythe University of Central Oklahoma (Applicant), and now U.S. Pat. No.11,058,521 B2, entitled “Method and apparatus for improvingosseointegration, functional load, and overall strength of intraosseousimplants” which application is a continuation-in-part and claims benefitof U.S. patent application Ser. No. 16/248,122 filed Jan. 15, 2019 bythe University of Central Oklahoma (Applicant), entitled “Nanofibercoating to improve biological and mechanical performance of jointprosthesis” the entire disclosure of which is incorporated herein byreference in its entirety for all purposes. This application claims thebenefit of U.S. Provisional Patent Application No. 62/634,993 filed onFeb. 26, 2018 in the name of Vagan Tapaltsyan and Morshed Khandaker,which is expressly incorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number5P20GM103447 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to the field of polymer fiberproduction and biomedical applications thereof. More specifically, theinvention relates to improving performance of metallic dental implantsby attachment of augmented electrospun fibers exhibiting micron to nanosize diameters.

BACKGROUND OF THE INVENTION

Polycaprolecton (PCL) Electrospun Nanofibers (ENF) have numerousbiomedical applications. Co-pending application Ser. No. 14/734,147 andU.S. Pat. No. 9,359,694 by the present Applicant discloses a method andapparatus for controlled deposition of branched electrospun fiber onbiomedical implants and material, the disclosures of which areincorporated herein by reference in the entirety. Research has shownthat micron to nano size fibers may be fused with biomedical implantsfor improving the mechanical and biological adhesion of titaniumimplants with the host tissue. Nano size fibers have been found to beexcellent carriers of drugs for improving bone growth. If applied as acoating around the implant, improved bone growth may reduce the implantloosening problem. U.S. Pat. Nos. 10,206,780 and 9,809,906 by thepresent Applicant disclose methods to achieve adhesion of functionalnanofiber coatings on a biomedical implant surface to increase theosteoinductive properties, and thereby to improve osseointegration of animplant, the disclosures of which are incorporated herein by referencein the entirety. The method uses PCL ENF fiber applied as a coatingmaterial, forming an extracellular matrix on an implant to improve ENFfiber adhesion with an implant surface, enabling use at physiologicalload bearing conditions. The method supports attachment of ENF fibers toan implant surface for both regular and irregular shape implants andenables drug delivery to promote bone growth.

The loss of teeth is a significant public health issue, increasing therisk of a wide range of conditions such as malnutrition due to lack ofproper masticatory function and clinical depression due to the change infacial appearance. Tooth loss is primarily caused by periodontaldisease, dental caries, or trauma. The prevalence of all risk factors oftooth loss increases with age and is thus projected to increase with thegrowth of the aging population across the world. In the United States,the population over 65 years of age is projected to reach 83.7 millionby 2050. Though the success rate of dental implant surgery is high, thefailure of implants due to poor osseointegration has been reported.

Threaded endosseous devices with a cylindrical or tapered shape are themost widely used type of dental implant. Endosseous dental implants aresurgically inserted into the jawbone. Osseointegration refers to bonegrown right up to the implant surface without interposed soft tissuelayer. Alveolar bone osseointegrates with the implant withoutdevelopment of a periodontal ligament. In cases of decreased primarystability of the implant in bone, micro-motions occur at the implantsurface that lead to osteoclast-driven resorption of bone around theimplant, contributing to further implant loosening and eventual implantfailure. Delayed bone healing leads to potential failure of the dentalimplant. Along with physical pain and suffering, implant loosening dueto poor osseointegration and healing leads to economic burdens.

Healing, surgical success, and complete osseointegration are regarded asthe most important characteristics of dental (and, to large extent,orthopedic) intraosseous implants. Currently, efforts to improve implantsuccess and osseointegration rates focus on the mechanical aspects ofimplants, such as the type of alloy, taper, screw thread design, metalfinish (acid and laser etching, polishing), etc. All intraosseousdevices of the above classes are classified as biologically inertimplants, as implant integration occurs through a process of boneremodeling, resulting in total ankylosis. Another approach to improveosseointegration is the direct attachment of osteoinductive nanoscaletopographies on endosseous dental implant surfaces. The main concernrelated to coating nanoscale materials onto an implant surface is therisk of coating detachment and toxicity of related debris. Further,implant length and diameter are important in determining the stabilityof the implant and the maximum load that can be placed on the implant.Generally, at least 7-9 mm of bone depth is required for implantplacement, with implant width varying from 3-7 mm in diameter. Thesedimensions often act as limiting factors when insufficient bone ispresent for implant placement. Thus, there is a need for strongerimplants with higher functional loads and osseointegration.

SUMMARY OF THE INVENTION

The present invention is directed to increasing success rate of implantsurgeries and decreasing integration time independent of the physicalproperties of implants. The present invention is unique in that itprovides a method of incorporating a system of bioaugmentation of metalimplants by introducing a PCL ENF carrying a recombinant growth factor,such as BMP2 (Bone Morphogenetic Protein 2) or PDGF (Platelet DerivedGrowth Factor). These growth factors have been extensively shown toactivate neural crest-derived bone stem cells to aid in differentiation,regeneration, and maturation of bone. Moreover, the properties of thefiber allow for binding to an antibiotic agent, such as silvernanoparticles (Ag NP), thus making the implant itself to exhibitantiomicrobial properties, decreasing the risk of implant failure due toinfection.

The aforementioned innovations can result in higher stability andretention of intraosseous implants. Thus, while current dental bio-inertimplants require 7-9 mm of bone depth, addition of modified PCL ENFs maydecrease that requirement due to growth factor-driven improved bonequality and improved osseointegration. Also, current bio-inert implantsrequire 4-6 months for complete osseointegration, while PCL ENFmodification may significantly reduce the osseointegration time andspeed up recovery. Finally, the present invention may allow for a widerrange of acceptable surgical sites for implant placement due to theENF's ability to stimulate new bone growth.

The present invention enables modification of an intraosseous implantdevice that is not only biologically non-inert, but can (1) stimulatebone and vascular growth (2) decrease localized inflammation, and (3)fight local infections. The method of the present invention provides afiber with at least any of the following modifications:

1. Nanofiber with PDGF

2. Nanofiber with PDGF+BMP2

3. Nanofiber with BMP2 and Ag

Further, nanofiber can be modified with many growth factors that havebeen shown to play a role in regulation of physiological bone remodelingto improve bone growth and maturation. These may include, for example,IGFs, TGF-β, FGFs, EGF, and WNTs, as well as BMP and PDGF. Bonedevelopment, remodeling, and repair requires attraction of mesenchymalprogenitor cells (MPC) and differentiation of MPC into osteoblasts. Theeffect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteinsfor primary human MPC has been shown to be highly significant. Thus, BMPand PDGF are the most commonly ones employed.

Titanium-based implants have been widely used in orthopedics andorthodontic surgeries because of their strong mechanical, chemical andbiological properties. We have tested a set of steps (e.g. grooving andoxidizing) by which a nanofiber matrix (NFM), composed of collagen (CG)and poly-ε-caprolactone (PCL) electrospun nanofibers, can be coated on aTi implant without subsequent detachment. A significantly improvedosseointegration of CG-PCL NFM-coated Ti over non-coated Ti notpreviously known was observed in our experiments. The advantage offunctional coating treatment on an implant is that it is simple,indirect, scalable, inexpensive, and supplementary to other surfacetreatment techniques. Such treatment can be applied on an implantsurface without affecting other implant factors, such as mechanical,medication (e.g. drugs, irradiation), and patient (e.g. age, osteopenia)factors. The biological properties of a functional coating can befurther improved by adding growth factors, proteins, and other moleculesto create a truly osteoinductive platform at the implant/bone interface.

In one major aspect the present invention provides an improvedintraosseous implant device capable of decreasing the time periods forosseointegration and preventing post-operative local infections.Titanium (Ti) alloy is most widely used as a dental implant material. Wehave developed a novel method of coating cylindrical Ti implants withnanofiber mesh by microgrooving. We have also immobilized fibronectin(FN), a glycoprotein of the extracellular matrix, on a Ti alloy(Ti-6Al-4V) by tresyl chloride-activation method. Our studies show thatmicrogrooving on cylindrical Ti implants and subsequent coating of thegrooves with collagen-poly-ε-caprolactone nanofiber matrix (CG-PCL NFM)significantly improves the biocompatibility, mechanical stability andosseointegration of Ti. A laser pulse can create microgrooves on thesurface of regular- and irregular-shaped implants. A literature searchhas revealed no reported research directed to the controlled fabricationof microgrooves on a complex-shaped implant surface, such as a dentalimplant (FIG. 1). The effect of coating the laser-induced microgrooveswith bone morphogenetic protein-2 (BMP2)- and silver (Ag) nanoparticle(NP)-immobilized PCL NFM on a dental implant has not been reported.Attachment of BMP2 and Ag NP onto Ti dental implant is sought via FN andCG immobilized PCL NFM, respectively. The above mentioned surfacetreatments on a dental implant may have the potential to improve theimplant osseointegration, reducing both healing time, and risk ofinfection.

In another major aspect the present invention provides methods forattachment of osseointegration-promoting and anti-bacterial biomoleculeson a dental implant using laser-induced microgrooves and PCL NFM.Immobilization of BMP2 with PCL NFM (referred as BMP2-PCL) andsubsequent coating of a laser-microgrooved titanium implant by BMP2-PCLmay lead to greater in vitro and in vivo osteogenic functions incomparison to the non-treated implants due to higher biologicalcompatibility of the BMP2-PCL-coated implant. Immobilization of Ag NPwith PCL NFM (referred as Ag-PCL) and subsequent coating of alaser-microgrooved titanium implant by Ag-PCL may lead to lower risk ofbacterial development in comparison to the non-treated implants due tohigher anti-bacterial resistance of the Ag-PCL-coated implant.

In another major aspect the present invention provides methods forfabrication of the control microgrooves at the interspace between twothreads of a dental implant. Fabrication of such grooves can havemedical benefits, since grooves on implants induce a higher amount ofimplant-bone contact area and osteoblast cell function in comparison toimplants without grooves. During implantation, the implant body isstrongly torqued and drilled into hard bone. The microgrooves protectthe functional NFM coating from these applied loads. The NFM coating canserve as a reservoir at the microgrooves on dental implant surfaces forcontrolled release of bone growth factor and anti-bacterial moleculesfor reducing infection and promoting osteogenesis. Laser pulse is amethod used to produce high precision, high roughness, and uniformmicrogroove topography on a dental implant along the threaded andnon-threaded sections.

In another major aspect the present invention provides methods forattachment of the functional PCL NFM coating on a dental implant of anysize or shape. Tresyl chloride, a chemical activation technique, can beused to attach FN on the dental implant surface directly. The bonegrowth signaling factors and collagen binding domain of FN can beutilized to attach bone growth factors (BMP2) and anti-bacterialmolecules (Ag NP) immobilized PCL NFM with Ti. The combined effect of FNimmobilization on laser-microgrooved Ti and coating those grooves by PCLNFM provides a novel technique to attach the functional PCL NFM coatingon any Ti implant.

In another major aspect the present invention provides methods for adirect immobilization technique using bone growth factors (BMP2) andanti-bacterial molecules (Ag NP) on a human dental implant surface viaPCL NFM. A potential opportunity for the advancement of in-vivotissue-to-implant osseointegration, faster healing times, and reductionof infection of a dental implant are possible from these inventions.Such treatment methods can be applied not only to improve dentalimplant-to-bone interface, but also to anchor many other orthopedicbiomaterials.

Nanofiber can be applied on the surface of an implant in several ways.First, a spiral micro-notching can be applied on the implant in the samedirection as the threads, with the nanofibers embedded into the notches.Second, the entire surface of the implant may be coated with a mesh ofnanofibers. Third, it can be a combination of both embedding andnotching.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a non-limiting diagram showing microgrooves andnanofiber-assisted drug delivery on dental implant as provided by thepresent invention.

FIG. 2 is a non-limiting diagram showing the method of the presentinvention providing protein immobilization on Ti using nanofiber matrixas a functional coating.

FIG. 3 is a non-limiting diagram showing F1s, S2p, N1s and O1s spectraof the Ti, Tresyl/Ti, and FN/Ti surface by a XPS analysis.

FIG. 4 is a non-limiting diagram showing precision microgrooves on theflat surface of a Ti rod formed by the method of the present invention.

FIG. 5 is a non-limiting diagram showing schematic representation of theprocesses for preparing an in vivo dental implant.

FIG. 6a is a non-limiting diagram showing a 3 mm diameter screw coatedwith 18 layers of PCL NFM.

FIG. 6b is a non-limiting diagram showing twisting of an NFM coatedscrew in to a pre-drilled hole (2.6 mm diameter) on a clear acrylicshows homogenous distribution of fiber along screw/acrylic interface.

FIG. 7a is a non-limiting image showing individual immobilization of CGand FN on PCL NFM.

FIG. 7b is a non-limiting graph showing that the individualimmobilization of CG and FN on PCL NFM has no adverse effect onosteoblast cells adhesion and proliferation of PCL NFM, and significantincrease of cell adhesion observed for FN-PCL-NFM when compared to PCLNFM (p<0.05).

FIG. 8a is a non-limiting graph showing a gradual increase of release ofBMP2 for 28 days was observed for FN-Hep-BMP2/PCL samples.

FIG. 8b is a non-limiting image showing cell divisions after 48 hours ofcell culture on FN-BMP2-PCL samples.

FIG. 9a is a non-limiting image showing PCL samples after Gram staining.

FIG. 9b is a non-limiting image showing CG-Ag-PCL samples after Gramstaining.

FIG. 10 is a non-limiting diagram presenting the test results oftime-dependent bone growth around the CG-PCL NFM-coated Ti implant andrelated in vivo pull-out tests to demonstrate mechanical stability ofmicrogrooved-Ti.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Our research demonstrates: (1) immobilization of ECM proteins (CG andFN) and bone growth factors (BMP2) with PCL NFM is possible, and suchimmobilization improves the in vitro cell viability of PCL NFM; (2)immobilization of antibacterial nanoparticles (Ag) with PCL NFM ispossible, and such immobilization improves the in vitro antibacterialactivity of PCL NFM; (3) direct attachment of FN on a dental implantmaterial (Ti-6Al-4V) is possible using tresyl chloride activationmethod; and (4) microgrooving of a Ti implant followed by coating themicrogrooves with CG-PCL NFM significantly improves in vivo mechanicalstability and osseointegration.

Referring to FIG. 1, in a preferred embodiment the present invention 10provides coating methods described above to dental implants with thegoal of improving the osseointegration of implants. We use a laser pulseto create microgrooves 11 at the interspace between two threads 18 of adental implant (Di) 12. Laser-induced microgrooves 11 were shown in ourresearch to significantly influence the surface morphology, contactangle, surface roughness, and chemical composition of Ti that caninfluence the attachment of fibronectin on implants. A set of continuousmicrogrooves 11 with 50 μm width, 5 μm depth, and 150 μm spacing betweengrooves are engraved at the root of the threads (˜0.5 mm) on a Di 12using a laser system (e.g., a Galvo FP fiber marking) to produce alaser-microgrooved Di 13. A rotary stage of the laser system (not shown)is oriented according to the helix angle of the implant threads toproduce the laser microgrooves 11 at the root 14 of the threads 18.

Referring to FIG. 2, in a preferred embodiment FN is attach on thelaser-engraved implant 12 surface by tresyl chloride method 21, andcoats the FN-immobilized implant surface 22 with BMP2- andAg-immobilized PCL NFM as shown 23. Basic terminal hydroxyl groups of apure titanium surface 22 react with tresyl chloride, which allows forfurther coupling with fibronectin (FN). Previous in vivo studies using arabbit femur model found that immobilizing fibronectin (FN) ontocylindrical pure titanium implants enhanced bone regeneration aroundimplants. However, pure titanium has limited applications in thebiomedical industry due to its inferior mechanical and biologicalproperties, compared to biomedical grade titanium alloys, such asTi-6Al-4V (the most commonly used titanium alloy in medical devices). Weexamined whether human plasma FN can be attached to Ti-6Al-4V via thetresyl chloride activation method. Three groups of samples were preparedto test the FN attachment on Ti via the tresyl chloride activationprocess: (1) control, (2) tresyl chloride-activated Ti (referred to asTresyl/Ti), and (3) tresyl chloride-activated Ti subsequently coupledwith FN (referred to as FN/Ti). To prepare Tresyl/Ti, the top surface ofa polished Ti-6Al-4V sample was treated with2,2,2-Trifluoroethanesulfonyl chloride at 36° C. for 48 hours, thenwashed with water, water-acetone (50:50), and acetone. Samples were thendried and stored in a desiccator. To prepare FN/Ti, a Tresyl/Ti samplewas treated for 24 hours at 37° C. with human plasma fibronectin dilutedin phosphate-buffered saline (PBS) solution to a concentration of 0.1mg/mL. X-ray photoelectron spectroscopy (XPS) analysis was conducted onall samples to determine the chemical state of Ti. The binding energyfor each spectrum was calibrated against the C1s peak at 284.8 eV.

Referring to FIG. 3, XPS analysis found the presence of an amide groupfor FN/Ti, which confirms the surface activation by tresyl chloride andthen direct coupling of FN with Ti. The N1s peak, derived from the amidebond of immobilized fibronectin, was detected around the binding energyof 400 eV for only FN/Ti samples. Therefore, this study suggested thatdirect attachment of FN is possible on a tresylated Ti alloy surface.Our proof of concept for the potential application of the treatmentprotocols on a Di led to the following: an ideal functional coating fora Di must reabsorb with time to allow and encourage new bone formationwhile maintaining its osteoconductive properties in vivo.

Referring to FIG. 4, the present invention provides laser engraving 41to support attachment of FN on a dental implant (FIG. 1-12) and thencoating the implant with BMP2 and Ag NP-immobilized PCL NFM. A laserpulse can be applied on the polished surfaces of Ti to create linear andcontinuous microgrooves on Ti implants. A laser capable of producingprecision microgrooves (FIG. 1-11) on at least the flat surface of a Tirod 42 can be used for this purpose. In our research we have used aGalvo FP fiber marking laser equipped with software for engraving a setof microgrooves (10 μm width, 5 μm depth, and 50 μm spacing betweengrooves) on Ti. The reason for achieving 5 μm-deep microgrooves on Ti isdue to the fact that each groove of this size can accommodate at least18 layers of nanofiber (average fiber diameter ˜300 nanometers). In thepresent invention, we use 18 layers of PCL NFM because our researchshows that the porosity of a PCL NFM membrane comprising 18 layers offibers is adequate for cells to migrate through the membrane. FN can beattached on laser engraved Ti implants (lgTi), where immobilization ofFN with Ti is accomplished by the tresyl chloride method as provided bythe present invention.

Referring to FIG. 5, aligned unidirectional PCL NFM can be collectedusing the methods disclosed in U.S. Pat. No. 9,809,906 by the presentApplicant and illustrated in FIG. 5. The laser-grooved surface of lgTiis activated by tresyl chloride and then 18 layers of PCL NFM isdeposited along the direction of the thread (clockwise) by rotating thetresylated Di 18 times until the implant collects 18 layers of fibers.The reason for adapting this coating method on a Di surface is due tofact that such a method should be able to maintain nanofibers along theDi/bone interface. FN, FN-Hep-BMP2 and CG-Ag complexes are gentlysplashed on the PCL coated Di samples to prepare FN-PCL/Di,FN-BMP-PCL/Di and CG-Ag-PCL/Di Di, respectively. All implants areprepared under sterile conditions and kept for 30 minutes in a portableultraviolet sterilizer before surgery.

Referring to FIGS. 6a and 6b , in a method validation test we coated aM3×0.5 screw by PCL NFM using the method of the present invention (FIG.5). We torqued the fiber-coated screw in to a pre-drilled hole (2.6 mm)on clear acrylic (FIG. 6b ). We observed homogeneously-distributed fiberalong the interface between the screw and the acrylic (FIG. 6b ).

EXPERIMENTAL ASPECTS Immobilization of Bone Morphogenic Protein-2 (BMP2)on Ti Using Fibronectin and PCL NFM.

Bone morphogenic proteins (BMPs) play important roles in in osteoblastand chondrocyte differentiation. Research shows that surfacefunctionalization of Ti with BMP2 improves the osteoblast activities ofTi. Among BMP family members, BMP2 is a potent osteoinductive factorthat plays key role during bone formation. Fibronectin (FN) is amultifunctional protein most abundantly found in the extracellularmatrix (ECM) under dynamic remodeling conditions such as bone healingand development. Research shows that tethering of FN onto Ti effectivelyenhanced the bone regeneration around implants. Our preliminary studiesshow that FN-immobilized PCL NFM (referred as FN-PCL) has higherbiocompatibility with osteoblast cells in comparison to PCL. FN containsbinding domains for many bone growth signaling factors, including BMP2and transforming growth factor-beta (TGF-β). We have successfullyimmobilized BMP2 with PCL NFM using FN in our preliminary studies. Theeffect of BMP2-immobilized PCL NFM coating on the osteogenic functionsof Ti is not known and thus it needs to be investigated.

Immobilization of Silver Nanoparticles (Ag NP) on Ti Using Collagen andPCL NFM.

Prolonged anti-bacterial activities of an implant are possible bytethering anti-bacterial molecules with the implant. Many studiesreported that Ag NP inhibits bacterial growth, while retaining/promotingosteoblast viability. Among common antibacterial nanoparticles (Ag, CuO,ZnO), Ag NP shows the minimum toxicity to environmentally relevant testorganisms and mammalian cells in vitro and in vivo. Since Ag NPdissolves in CG, it can be immobilized with CG-PCL NFM. Our in vivo andin vitro studies show that CG-PCL NFM coating enhanced biologicalfunctions of Ti. This is due to the fact that higher cell functions werecreated via better cell signaling arising from the cell-cell contact andthe cell-NFM components in the case of the CG-PCL NFM-coated Ti samplesthan non-coated Ti samples. Our preliminary studies showed noantimicrobial activity of Ag NP-immobilized CG-PCL NFM towardsStaphylococcus aureus in comparison to PCL NFM. The effect of AgNP-tethered CG-PCL NFM on the osteogenic and anti-bacterial activitiestowards other common aerobic bacterial organisms on Ti implant is notknown and thus needs to be investigated.

Effect of Immobilization of Fibronectin and Collagen on the CellularFunctions of PCL NFM

Fibronectin (FN) contains several active sites, known as theheparin-binding domains, collagen-binding domain, fibrin-binding domain,and cell-binding domain, that serve as platforms for cell anchorage. Thegoal of this preliminary study was to evaluate the effect ofimmobilization of collagen and plasma fibronectin with PCL NFM on thecellular functions of PCL NFM. The results (FIG. 7a and FIG. 7b ) showthat the individual immobilization of CG and FN on PCL NFM has noadverse effect on osteoblast cells adhesion and proliferation of PCLNFM, although a significant increase of cell adhesion was observed forFN-PCL-NFM when compared to PCL NFM (p<0.05). A significant improvementof cell adhesion and proliferation was observed for FN-CG-PCL NFM incomparison to PCL NFM (p<0.01). This is due to the fact that higher cellfunctions were created via better cell signaling arising from thecell-cell contact and the cell-NFM components in the case of FN-CGimmobilized PCL NFM compared to PCL NFM.

Direct attachment of FN on a Ti implant surface is possible using aTresyl Chloride-Activated Method (shown in Section C.5.). Since FNcontains a CG binding domain, FN-immobilized Ti can therefore bepolymerized into CG-PCL. The effect of the attachment of PCL NFM with Tiusing CG and FN on the osteogenic functions of the implant is not knownand needs to be investigated.

Immobilization of Human Bone Morphogenic Protein-2 (BMP2) with PCL NFMUsing Fibronectin (FN).

The PCL NFM can be modified with heparin (Hep) and further immobilizedwith BMP2. The modified fibers showed the potential to effectivelyinduce osteogenic differentiation of periodontal ligament cells. SinceFN contains heparin-binding domains, PCL fibers can be modified withFN-Hep-BMP2 complex. The purpose of this preliminary study wasthreefold: (1) to immobilize BMP2 on PCL NFM using only FN-BMP2 andFN-Hep-BMP2 complexes, (2) to determine the amount of BMP2 release fromthe immobilized BMP2-PCL NFM, and (3) to compare the cell viability ofBMP2-immobilized PCL NFMs with respect to PCL NFM (control). ImmobilizedBMP2 was released from the PCL NFMs in a sustained manner for 28 days,although the rates of release of BMP2 from FN-BMP2/PCL andFN-Hep-BMP2/PCL were different. A gradual increase of release of BMP2for 28 days was observed for FN-Hep-BMP2/PCL samples (FIG. 8a ). Rapidrelease of BMP2 for first 4 days, then gradual decline of release ofBMP2 with time was observed for FN-BMP2/PCL samples. Cells displayed awell-extended morphology on all the BMP2-treated groups, when they werecompared with the control group (8 a). FIG. 8b depicts a representativeimage showing cell divisions after 48 hours of cell culture onFN-BMP2-PCL samples. In the image, blue color shows the attachment ofcells on NFM. There was more than a 52% and 30% increase in the cellviability on FN-Hep-BMP2-PCL samples after culturing the cells for 72hours compared to control and FN-BMP2-PCL. Both release and cellviability tests suggested an advantage of FN-Hep-BMP2 over FN-BMP2complex for the immobilization of BMP2 with PCL NFM. FN-Hep-BMP2immobilized PCL NFM has the potential to induce osteogenicdifferentiation of osteoblast cells on a Ti implant surface, which isnot yet known. The effect of the treatment of Ti with FN by tresylchloride method and then coating by FN-Hep-BMP2/PCL on the osteogenicfunctions needs to be investigated.

Attachment of Silver Nanoparticles (Ag NP) with PCL NFM Using CG

Silver nanoparticles (Ag NP) show promising anti-bacterial propertieswith biocompatibility and minimal toxicity. Ag NP-loaded collagen wasimmobilized with polymeric film to inhibit bacterial growth whilepromoting osteoblast cell viability. The anti-bacterial activities ofPCL NFM can be improved by immobilizing Ag NP-loaded CG with PCL NFM.The purpose of this preliminary study was to examine the effect ofimmobilization of Ag NP-loaded CG on the anti-bacterial properties ofPCL NFM. We succeeded in immobilizing Ag-loaded collagen with PCL NFM.The SEM and XRD analysis before and after 2 days of bacterial cultureconfirmed the presence of Ag with PCL. Our bacterial culture studiesshowed no sign of colonies growing on Ag-CG-PCL, whereas the presence ofbacteria was observed in PCL. FIG. 9 shows PCL samples after Gramstaining: (a) PCL and (b) CG-Ag-PCL. S. aureus that take up the Gramstain were present in PCL, as observed in the image by circular blackshapes (pointed by arrows), while CG-Ag-PCL without S. aureus, did notstain and appears with a gray color. One reason for this might arisefrom an increased carrying capacity of Ag NP-loaded collagen by thenanofiber disc due to its unique surface-to-volume ratio.

In Vivo Evaluation of Coating a Titanium Implant with CG-PCL NFM

We have invented a method of coating a cylindrical metal implant withNFM that is made with CG-PCL (U.S. Pat. No. 9,809,906). Our inventionimplements a set of grooves that are created on Ti in a circumferentialdirection to increase the contact area between the implant and bone.CG-PCL NFM is subsequently coated along the sub-micrometer grooves onthe Ti implant using our unique electrospinning process (U.S. Pat. No.9,359,694). The goal of this research was to evaluate the effect ofCG-PCL NFM coating on the mechanical stability and osseointegration of aTi implant using a rabbit model. Our in vivo pull-out tests demonstratedthat mechanical stability of microgrooved-Ti was significantly highercompared to non-grooved Ti. The mechanical stability (quantified byshear strength) of groove-NFM Ti/bone samples were significantly greatercompared to other samples (p<0.05). The pull-out strength ofgroove-NFM-coated Ti was comparable to other functional coating-treatedTi reported in the literature. The types of new bone growth on Ti wasdifferent between groove and groove-NFM samples, which was observed fromthe stained images of histology-sectioned images (FIG. 10e ).Histomorphometric results showed that the amount of BIC on Ti was higherfor groove-NFM (12.18±0.94 mm, n=2) than groove (5.30±4.01 mm, n=2)samples. Due to the poor attachment of Ti with bone, Ti implants cameout from their implant sites during histology preparation. Therefore,there was no result for any control sample. Both μCT analyses (FIG. 10eand FIG. 10f ) and blood serum (FIG. 10g ) results confirmed thetime-dependent bone growth around the CG-PCL NFM-coated Ti implant anddetermined that 6 weeks were required for sufficient bone growth aroundthe implant.

Our in vivo pull-out tests demonstrated that mechanical stability ofmicrogrooved-Ti was significantly higher compared to non-grooved Ti(FIG. 10c ). The mechanical stability (quantified by shear strength) ofgroove-NFM Ti/bone samples were significantly greater compared to othersamples (p<0.05). The pull-out strength of groove-NFM-coated Ti wascomparable to other functional coating-treated Ti reported in theliterature. The types of new bone growth on Ti was different betweengroove and groove-NFM samples, which was observed from the stainedimages of histology-sectioned images (FIG. 10e ). Histomorphometricresults showed that the amount of BIC on Ti was higher for groove-NFM(12.18±0.94 mm, n=2) than groove (5.30±4.01 mm, n=2) samples. Due to thepoor attachment of Ti with bone, Ti implants came out from their implantsites during histology preparation. Therefore, there was no result forany control sample. Both μCT analyses (FIG. 10e and FIG. 10f ) and bloodserum (FIG. 10g ) results confirmed the time-dependent bone growtharound the CG-PCL NFM-coated Ti implant and determined that 6 weeks wererequired for sufficient bone growth around the implant.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the invention may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description of the invention.Changes may be made in the elements described herein without departingfrom the spirit and scope of the invention as described in the followingclaims.

1-9. (canceled)
 10. A threaded intraosseous implant, comprising: acylindrical device having external threads extending from a root of thedevice along a helix angle with interspaces located between turns ofsaid threads; grooved surfaces comprising at least one microgroove atthe interspaces between thread turns and oriented according to the helixangle of said threads; nanofibers coating said grooved surfaces, saidnanofibers substantially aligned with the helix angle of said threads,wherein, said at least one microgroove is adapted to mitigatephysiological loading of said nanofibers during insertion of saidimplant into biological bone.
 11. The threaded intraosseous implantaccording to claim 24, wherein said at least one microgroove exhibits acapacity to hold 18 layers of nanofibers.
 12. The threaded intraosseousimplant of claim 25, wherein said nanofibers are adhered to saidmicrogrooves.
 13. The threaded intraosseous implant of claim 26, furthercomprising tresyl chloride coupled with fibronectin (FN) coating saidgrooved surfaces;
 14. The threaded intraosseous implant of claim 24,wherein said intraosseous implant is adapted to attach a prosthesis tosaid biological bone.
 15. The threaded intraosseous implant according toclaim 24, wherein said intraosseous implant is one of a hollow or solidscrew device.
 16. A threaded intraosseous implant, comprising: acylindrical device having external threads extending from a root of thedevice along a helix angle with interspaces located between turns ofsaid threads; grooved surfaces comprising at least two microgrooves atthe interspaces between thread turns and oriented according to the helixangle of said threads; nanofibers coating said grooved surfaces, saidnanofibers substantially aligned with the helix angle of said threads,wherein, said at least two microgrooves are adapted to mitigatephysiological loading of said nanofibers during insertion of saidintraosseous implant into biological bone, and wherein said intraosseousimplant is adapted to attach a prosthesis to said biological bone. 17.The threaded intraosseous implant according to claim 30, wherein said atleast two microgrooves exhibit a capacity to hold 18 layers ofnanofibers where microgroove depth is 5 μm.
 18. The threadedintraosseous implant of claim 30, further comprising tresyl chloridecoupled with fibronectin (FN) coating said grooved surfaces.
 19. Thethreaded intraosseous implant of claim 30, wherein said nanofibersfurther comprise a nanofiber matrix (NFM) adhering to said groovedsurfaces.
 20. The medical intraosseous implant according to claim 30,wherein said intraosseous implant is one of a hollow or solid screwdevice.
 21. A threaded intraosseous implant, comprising: a threadedcylindrical device and having an external thread extending from a rootof the device along a helix angle with interspaces located between turnsof the thread; grooved surfaces comprising of two parallel microgroovesat the interspaces between the thread turns and oriented according tothe helix angle of said thread to provide a helix angle of themicrogrooves, tresyl chloride coupled with fibronectin (FN) coating saidgrooved surfaces; a nanofiber matrix (NFM) coating said groovedsurfaces, said NFM substantially aligned with the helix angle of saidmicrogrooves, wherein, said NFM is adhered to said grooved surfaces ofsaid intraosseous implant, and wherein, said microgrooves are adapted toat least mitigate physiological loading of said NFM.
 22. The threadedintraosseous implant according to claim 35, wherein said microgroovesexhibit a capacity to hold 18 layers of nanofibers where microgroovedepth is at least 5 μm.
 23. The medical intraosseous implant accordingto claim 35, wherein said NFM comprises 18 fiber layers, said layersdeposited circumferentially along the helix angle of said microgroovesand shielded from applied loads.
 24. The threaded intraosseous implantof claim 35, wherein said microgrooves are engraved to a depth of 5 μm,a width of 50 μm, and a spacing of 50 μm to 150 μm between said groovesat the interspace of said thread.
 25. The threaded intraosseous implantof claim 35, further comprising attached collagen loaded with silvernanoparticles (Ag NP) or antimicrobial analogs thereof.
 26. The threadedintraosseous implant of claim 35, wherein said NFM comprises at leastone of growth factor or antibiotic-modified polycapronlectron (PCL)Electrospun Nanofibers (ENFs), said PCL-ENF combined with at least anyof Platelet Derived Growth Factor (PDGF), Bone Morphogenetic Protein 2(BMP2), PDGF+BMP2, or BMP2 and silver (Ag).
 27. The threadedintraosseous implant of claim 35, wherein said intraosseous implant isadapted for insertion in biological bone, and further adapted to attacha prosthesis to said biological bone.
 28. The threaded intraosseousimplant according to claim 35, wherein said intraosseous implant furthercomprises Titanium or analogs thereof.
 29. The threaded intraosseousimplant according to claim 35, wherein said intraosseous implant is oneof a hollow or solid screw device.